Anal. Chem. 1995,67,1067-1073
Capillary Electrophoresis of Inorganic Anions in Nonaqueous Media with Electrochemical and Indirect UV Detection Hossein Salimi-Moosaviand R. M. C a d d y * Department of Chemistly, University of Saskatchewan, Saskatoon, SK, S7N OWO, Canada
Methanol and dimethylformamide were evaluated for the separation of a series of inorganic anions. As these solvents were added to an aqueous electrolyte, the electroosmotic flow went through a minimum at 70-80% (v/ v) organic solvent and then increased as the composition approached that of the pure solvent. Reproducibility of electroosmotic flow was good (%RSD = 1.1 for dimethylformamide), and Joule heating was not as important as in water systems. The effect of electrolyte concentration on electroosmotic flow was similar to that expected for aqueous solutions, but significant differences in separation selectivity were observed, with many ions showing reversed separation order compared to aqueous systems. In addition, there was some evidence of ion association effects that led to changes in selectivity as a function of concentration and nature of the electrolyte. The application of indirect W detection in methanol was briefly evaluated with chromate, phthalate, and benzoate electrolytes. Phthalate electrolyteswere slightly less sensitive than chromate, but chromate was not stable in methanol. Calibration of 11 inorganic anions separated in a phthalate electrolyte gave linear curves in the range of 5 x 10-5-8 x mol& detection limits were in the range of 2.0 x 10-5-3.4 x moVL. Amperometric detection at a 25 pm Pt disk electrode gave detection limits in the range of 1 x 10-9-6 x moVLfor some anions, but calibration curves were linear only over small ranges of concentration. Peak shapes obtained with the electrochemical detection systems were good, and theoretical plate counts were in the range of 140 000-450 000. Capillary electrophoresis (CE) is a powerful technique for separation of ionic and neutral sub~tances.~-3Since its first introduction by Jorgenson and Lukacs,4-'j new publications in this area have been growing at an exponential rate. Most CE separations of small ionic species have been performed in aqueous and for these species there are only a limited number of experimental parameters available for optimization of separation selectivity. The use of nonaqueous solvents in place of aqueous systems may offer advantages as a means for selectivity adjust(1) Jorgenson, J. W. Trends Anal. Chem. 1984,3,51-54. (2)Grossman, P. D.;Colbum, J. C.; Lauer, H. H.; Nielsen, R G.; Ripgin, R M.; Sittampalam, G. S.; Rickard, E. C. Anal. Chem. 1989,61,1186-1194. (3)Jones, W. R;Jandick, P.J. Chromatogr. 1992, 608, 385-393. (4)Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (5) Jorgenson, J. W.; Lukacs, IC D.]. Chromatogr. 1981,218,209-216. (6)Jorgenson, J. W.; Lukacs, K. D. HRCC CC,J. High Resolut Chromatogr. Chromatogr. Commun. 1981,4,230-231. 0003-2700/95/0367-1067$9.00/0 Q 1995 American Chemical Society
ment for many analysis situations. Since solvation of solutes should be considerably different in organic solvents, nonaqueous solvents may have profound effects on separation selectivity. The physical and chemical properties of nonaqueous solvents are also expected to be quite different from that for water and this should introduce additional flexibility for the optimization of separations. Some of the experimental parameters expected to be useful include a wider range of acid/base properties, ion pairing, increased solubility for solutes insoluble in aqueous solution, reduced sorption of hydrophobic substances (i.e., long-chain surfactants) onto capillary walls, and reduced Joule heating. For electrochemicaldetection, relatively large working potential ranges should be available. Although nonaqueous CE systems offer a number of potential advantages, there are some uncertainties associated with their application to CE. Appreciable ion pairing may affect peak shape and detection sensitivity. Purification of organic solvents is often difficult, and water, which is a common contaminant in organic solvents, may affect electroosmotic reproducibility and separation selectivity. In addition, volatility, viscosity, and toxicity are parameters that must be considered. Although nonaqueous media have been exploited in classical electrophoresis, their application in CE is virtually an unexplored area. Some of the classical electrophoretic studies in nonaqueous solvents have included measurement of the mobility of carbonblack particles in k e r ~ s e n eseparation ,~ of aqueous insoluble dyes and biological compounds in a variety of organic solvents? separation of alkali metal cations by paper electrochromatography in different organic solvents? separation of various anions and cations in methano1,lOJl and separation of lubricating oil additives and other organic c o m p ~ u n d s . ~ ~Korchemnaya -l~ et al. have reviewed these and other uses of nonaqueous media in classical electroph~resis.~~ Walbrohel and Jorgenson,16who were the first to exploit pure nonaqueous media, examined acetonitrile for the separation of quinolines. Most other published studies have looked at mixed aqueous/organic solvent systems, and of these there are only a limited number where the use of appreciable (7) Hayek, M. J. Phys. Colloid Chem. 1951,55, 1527-1533. (8)Paul, M.H.; D u m " E. L. J. Am. Chem. SOC.1952, 74,4721-4722. (9)Tuckeman, M. M.;Strain, H. H. Anal. Chem. 1960,32,695-698. (10) Beckers, J. L.; Everaerts, F. M. J. Chmmatogr. 1970, 51,339-342. (11) Beckers, J. L.; Everaerts, F. M. J. Chmmatogr. 1972, 68,207-230. (12)Leighton, D.;Moody, G. J.; Thomas, J. D.R Analyst 1974, 99,442-452. (13)Tshabalala, M.A;Scham, S. B.; Gerberich, F. G.; Lowman, D. W.; Rodgers, L. B.J Chromatogr. 1981,207,353-363. (14)Parekh, N.J.; Fatmi, A A; Tshabalala, M. A; Rodgers, L. B.J. Chromatog, 1984,314.65-82, (15) Korchemnaya, E. K; Emakov, A N.; Bochkova, L. P. J. Anal. Chem. USSR (Engl. Transl.) 1978,33,635-639. (16)Walbrohel, Y.;Jorgenson, J. W. J. Chromatogr. 1984,315,135-143.
Analytical Chemistty, Vol. 67,No. 6, March 15, 1995 1067
amounts of organic solvents is r e p ~ r t e d . ' ~Recently J~ CE aspects of current, voltage, and ionic strength have been reported for formamide.lg The aim of this work was to study the potential of nonaqueous media as an alternative electrolyte medium in CE. The main focus was on separation selectivity for small inorganic anions and electroosmotic flow behavior. Two different modes of detection were examined. First,indirect UV detection in nonaqueous media was examined because it would permit the detection of a wide range of test analytes and also because this technique is commonly used in aqueous systems. Second, the potential of electrochemical detection was briefly studied because of the possible benefit of wider working potential ranges with nonaqueous solvents. EXPERIMENTAL SECTION CE with Amperometric Detection. Polyimide-coated fused
silica capillaries, 25 pm i.d. and 370 pm 0.d. (Polymicro Technology, Phoenix, AZ), were used in lengths of 50 cm. The highvoltage power supply (30 kV, Spellman, Model CZE1000PN30R, Plainview, NY) was housed in a Plexiglas box with an interlock on the access door to protect the operator. The detection cell and potentiostat were placed in a faradaic cage to minimize interference from external sources of noise. The electronic circuit diagram of the potentiostat was given previously.2o Electrochemical detection with a threeelectrode system was controlled with a PCL818 high-performance data acquisition card (B&C Microsystem, Sunnyvale, CA). In-house computer software controlled the application of potential to the electrodes and the collection and display of the data; the computer software also included programs for cyclic voltammetry and pulsed amperometric detection. Ptdisk working ultramicroelectrodeswere prepared from 25 pm 0.d. Pt wire (99.9%, Goodfellow, Cambridge, UK), which was heat sealed in soft-glasstubing. This electrode was then polished with carborundum abrasive paper (Diamond, waterproof paper, No. 600)and 0.3 pm alumina. The reference electrode was a miniature saturated calomel electrode (SCE), and the auxiliary electrode was a Pt wire (500 pm 0.d.). A capillary was k e d in place over the top of a 0.5 mL polyethylene vial that was attached to a platform that also had a XYZ micropositioner (Model MR3, Klinger, Garden City, NY) attached to it. The working ultramicroelectrode was fastened on to this positioner, and with the help of a microscope for both top and side view directions, the electrode was positioned directly in front of the capillary exit at a distance of -10 pm. The reference and counter electrodes were placed into the polyethylene vial, and electrolyte, which was added to cover both the capillary and the electrode, was held above the top of the vial by surface tension. Samples were introduced by electromigration at 1kV for 10 s. Samples of iodine (0.001 mol/L in dimethylformamide) were used for electroosmotic flow measurements; detection was at -1000 mV vs SCE. All other anions were detected at 1700 mV vs SCE. CE with W Detection. A Quanta 4000 (Waters Chromatography Division of Millipore, Milford, MA) CE unit was used for (17) Benson, L. M.; Tomlinson, A. J.; Reid, J. M.; Walker, D. L.;Ames, M. M.J. High Resolut. Chromatogr. 1993,16,324-326. (18)Tomlinson, A. J.; Benson, L. M.: Naylor, S. J. High Resolut. Chromatogr, 1994,17, 175-177. (19) Sahota, R; Khaledi, M. G. Anal. Chem. 1994, 66,1141-1146. (20) Lu, W.; Cassidy, R. M.; Baransky, A S. J. Chromutou. 1993,640, 433440.
1068 Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
UV detection (254 nm). Capillaries were 75 pm i.d. and 370 pm 0.d. (Polymicro Technology), with a length of 65 cm and an endto-detection length of 58 cm, unless specified otherwise. Samples were injected hydrostatically by elevation of the sample vials to 10 cm for 15 s. All samples were dissolved in water. The tip of the water peak was used as a neutral marker for the electroosmotic flow measurements. Anions were separated in 0.01 mol/L potassium hydrogen phthalate (KHP), 0.02 mol/L n-butylamine (a-BuNHz), and 2% (v/v) water in methanol. Capillaries were washed for 1 min between runs by application of a 15 mmHg vacuum. Chemicals. All sample solutions were prepared from deionized water that was double distilled. The background electrolytes for separation and electrochemical detection were tetrabutylammonium hexafluorophosphate (TBAHP Sigma Chemical Co., St. Louis, MO) and tetraethylammonium perchlorate (TEAP), which was prepared by a slow and careful addition of HC104 (70%v/v) to tetraethylammonium hydroxide (20% w/v in water, Sigma Chemical Co.); the salt was recrystallized in water and washed with methanol. Dimethylformamide, n-BuNH2, methanol, and acetonitrile were reagent grade. Stock solutions of (0.01 mol/L) sodium fluoride, sodium chloride, sodium bromide, sodium iodide, sodium azide, sodium nitrite, sodium nitrate, sodium sulfate, sodium thiosulfate, sodium oxalate, and potassium thiocyanate (all reagent grade) were prepared in deionized water, and samples were diluted to the desired concentration either with deionized water or the solvent present in the separation electrolyte. For electroosmotic flow measurements, solvents were dried with a molecular sieve (Type 5 4 MC&B, Los Angeles, CA). A 0.001 mol/L solution of iodine in dimethylformamide was used as neutral marker for electroosmotic flow measurements. For indirect UV detection, the stock background electrolytes were sodium chromate (Aldrich, Milwaukee, WI), sodium benzoate (BDH, Toronto, ON) (1mol/L), and KHP (BDH) (0.5 mol/L) in water. These electrolytes were diluted to 0.01 mol/L with methanol. All electrolytes and samples were filtered through a 0.2 pm Nylon-66 membrane syringe filter (Cole-Parmer, Chicago, IL) immediately prior to use. Procedures. In both electrochemical and W detection systems, the applied separation potential was -30 kV (detector at anode end), and for electroosmotic flow measurements, the potential was +30 kV (detector at cathode end). Capillaries were conditioned by washing with solvent for 1h, followed by another 2 h with the separation electrolyte by the use of 10 cm height differential. Some capillaries were also conditioned with NaOH and HC1 solutions, but this had no noticeable effect. All glassware was rinsed with a chromic acid/sulfuric acid solution followed by water and acetone and then dried in an oven. The number of theoretical plates was calculated from, N = 5.54(tR/w0.32, where t~ is the migration time of analyte and ~ 0 . is 5 the peak width at half-peak height; the values of efficiency were calculated to ensure that there were no major problems as a result of slow ion association equilibria or sorption effects. Electroosmotic mobilities were calculated from peo= U/(teoV), where peois electroosmotic mobility, teois the migration time for the neutral marker (in s), V is the separation voltage (in V), L is the capillary length, and 1 is end-to-detection window length (in cm), The electrophoretic mobilities of anions were calculated from pep= peo + U / ( t e p v , where peo and pepare in opposite directions; the direction of electroosmotic flow was from anode (+) to the cathode (-),
while the anions moved toward the anode. The linearity of calibrationcurves for anions was evaluated from plots of sensitivity vs concentration.21The sensitivity, S,was obtained from, S = (I - b)/C, where I and C are the peak-area responses and concentration, respectively, and b is the Y-axis intercept obtained from the analysis of least-squares regression using the original response/ concentration dahz1
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RESULTS AND DISCUSSION Choice of Organic Solvent Systems. Although, there is a wide variety of organic solvents with properties that could be suitable for CE, this study focused on dimethylformamide and methanol. Dimethylformamide was chosen because of its high dielectric constant, high boiling point, and the fact that it has been used extensively for electrochemical studies. Methanol was chosen mainly because it is a common solvent and easily accessible to analytical chemists. Except for certain specific experiments, solvents were not purified or dried. For CE analysis with electrochemical detection, TEAP and mAHP were selected as electrolytes mainly because they are common electrolytes used in nonaqueous electrochemical studies. Electroosmotic Flow in Organic Solvents. Control and characterization of electroosmotic flow is important in the evaluation of reproducibility and for peak identification. Consequently, the behavior of electroosmotic flow was examined for electrolyte solutions containing 0-100% of dimethylformamideand of methanol. Previous literature reports have described the effect of the addition of organic solvents to aqueous electrolyte^^^^ up to a maximum organic solvent content of 80% (v/v) ,and extrapolation of these results suggests that electroosmotic flow might be very small in pure organic solvents. Results in Figure 1show that as dimethylfomamide (curves 1 and 2) and methanol (curve 3) content increased to 75% (v/v), pm decreased, which is in agreement with trends observed in previous studies.22a However, further addition of organic solvent resulted in a signiscant increase in peo. The results in Figure 1 show that the direction of electroosmotic flow was from anode to the cathode, as for aqueous electrolytes, and that the magnitude of electroosmotic flow was slightly smaller or comparable to the electroosmoticflow observed in similar aqueous systems. Electroosmotic flow in mixed aqueous/nonaqueous media has been attributed to the dissociation of silanol groups and to specific adsorption of ions on the silica surface.n The physical parameters that influence electroosmotic flow are dielectric constant ( E ) , viscosity (v), and zeta potential (5). These parameters are related to pea by the Von Smoluchowski equation:24pe, = EocrC /4n7, where p m is the electroosmotic mobility and eo is the permittivity in vacuum (8.854 x 10-l2 C2J-' m-l). Kenndler et al.22have summarized their and other literature values of 47,and this compilation shows this ratio decreases upon the addition of organic solvent to give a minimum value between 60 and 80% v/v organic solvent. Addition of organic solvent can also affect the 5 potential at silica surfaces via an increase in the pKa of the silanol groups.22 An increase in pKa should lower the 5 potential, and this may contribute to the decreasing values of p,, from 0 to 75% (v/v) organic solvent (see (21) Cassidy, R M.; Janoski,M.LCGC 1992,IO, 692-696. (22) Shwer, C.; Kenndler, E. Anal. Chem. 1991,63, 1801-1807. (23) Fujiwara, S.;Honda, S. Anal. Chem. 1987,59,487-490. (24) Smoluchowski, M. Handbuch der Elecktrititat und des Mugnetismus; Lipng, Germany, 1914; Vol. 2, p 366.
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F i i e 1). The increase in pm above 75%(v/v) shows that changes in 4 7 must have a larger effect on the pw. To determine whether the Von Smoluchowski equation (see above) would adequately describe the experimental results in curves 1-3 of Figure 1,values of electroosmotic flow as a function of methanol content were calculated from the experimental value of electroosmotic flow in water and values of E / V ~given in the literature for methanol. The equation used was p e o ( n o m ) = peo(mter)[ ~ / ~ 5 1 c ~ a t e r ) / [ ~(mixture), /~CI where peo(water) was the experimental result from this study. The results of this calculation are shown in curve 4 of Figure 1, and it can be seen that this model shows reasonable agreement with the experimental results for methanol (curve 3). The consistently higher values of in methanol (curve 3) compared with calculated values (curve 4) may be due to increased ion association as a function of methanol content; any ion association should increase the 5 potential due to a decreased ionic strength. During initial studies with dimethylformamide the migration times of different neutral markers were found to shift randomly in the range of 238.7-281.5 s. Removal of trace water from the solvent and the use of multiple largevolume electrolyte reservoirs connected with diffusion tubes to help isolate electrolysis products had little effect on this shift in migration times. However, since the electrolytes were not buffered, it was felt that the composition of the double layer in pure organic solvents might be especially sensitive to very small amounts of electrolysis products like H+ and OH-. Measurements of pH for used (3 h period) and new electrolytes, both mixed to 50%(v/v) with water, gave pH values of 6 and 9, respectively. To overcome this change in pH, n-BuNHz (0.1 mol/L) was added to the electrolyte, and the %RSD for migration times improved to 1.1%(n = 9) over a 10 h period. The results in Figure 2 show that both the change in peawith amine concentration and the RSD values for p, stabilized quickly upon Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
1069
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Figure 2. Electroosmotic mobility as a function of concentration of rrBuNH2: separation electrolyte, 0.01 mol/L KHP and 2% (vh) water in methanol; 65 cm capillary (75p m i.d.) with end-to-detection point of 58 cm; separation voltage, -30 kV; %RSD shown as error bars.
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the addition of n-BuNHz. The addition of n-BuNHz first increased the electroosmotic flow, most likely a result of an increase in charge at the capillary wall through the dissociation of silanol groups,which is expected to occur once the KHP in the electrolyte was neutralized. It was also found that excess n-BuNHz was necessary for the separation of some inorganic anions such as oxalate, thiosulfate, sulfate, and fluoride. Ionic Strength Effects. In aqueous electrolytes an increase in ionic strength reduces the g potential, and consequently causes a reduction in electroosmotic flow. The results in Figure 3 show that decreases in electroosmotic flow were also observed for increased concentrations of both TEAP and TBAHP electrolytes in dimethylformamide. At lower concentration both TEAF'and TBAHP electrolytes produced similar values of p,, (see Figure 3), but at higher electrolyte concentrationp,, values in TBAHP were larger, possibly as a result of ion pairing in this electrolyte. This is contirmed by the fact that an essentially linear plot was 1070 Analytical Chemistry, Vol. 67, No. 6, March 15, 7995
observed for a polynomial fit of the TEAF' data, but the fit for TBAHP showed appreciable curvature (dotted lines in Figure 3 are polynomial fits). Since linear plots of electrophoreticmobility vs log(concentrations) are expected for ideal ionic solutions,25the curvature for TBAHP may be a result of ion pairing, but interactions between the silica and the TBA cation may also inhence the response in this system. Moderate amounts of ion association should produce specific ion/ion interaction that could be useful for adjustment of separation selectivity, and evidence for such effects is discussed below. Joule Heating Effects. The use of higher electric fields can reduce analysis time and enhance separation efficiency, but with aqueous media high ionic strengths can lead to appreciable Joule heating.26~2~This heating can have detrimental effects on the separation, especially for thermally sensitive compounds (i.e., protein denaturation) Joule heating effects have been examined in some detail for formamide,lgand in this present study Joule heating was briefly examined for methanol. The results in Figure 4 show that peowas essentially linear in methanol, whereas some curvature was observed at higher electric fields in aqueous solutions. These limited results tend to c o h conclusions made for formamide in previous studies,1gwhich are that Joule heating in organic solvents should not be a serious concern and that it may be possible to use higher electric fields than for aqueous systems. Selectivity. Figure 5 summarizes the selectivity patterns observed for 11anions in four solvent systems containii the same electrolyte. Relationships similar to those shown in Figure 1for electroosmotic flow are observed for each of these ions if plots of mobility vs percent methanol (these curves are not shown) are made. The order of the anions along the X-axis in Figure 5 is the same as the order of migration for separations in methanol (see Figure 6). The results in Figure 5 show that signiiicant changes in separation order were observed relative to that in aqueous systems, and for some ions a complete reversal in separation order was seen. For example, thiocyanate was the second fastest ion in methanol but the slowest in water, while SzO32- was the second slowest ion in methanol, but the fastest in water. For halogens the migration order in the methanol was iodide, bromide, chloride, and fluoride, while a reversed separation (25) VanOrman, B. B.; Liversidge, G. G.; McIntire, G. L. j . Microcolumn Sep. 1991,2, 176-180. (26) Rasmussen, H. T.; McNair, H. M.J. Chromatogr. 1990,516, 223-231. (27) Atria, IC D.; S i p s o n , C. F. Chromatographia, 1987,24, 527-532. (28) Nelson, R J.; Paulus, A; Guttman, A; Cohen, A S.; Karger, B. L. j. Chromatogr. 1989,480,111-128. (29) McCormick, R M. Anal. Chem. 1988,60, 2322-2328.
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Time (min) Figure 6. Separation and detection of inorganic anions with phthalate electrolyte: electrolyte, 0.01 mol/L KHP, 0.02 mol/L r+ BuNH2, and 2% (viv) water in methanol; indirect UV detection at 254 nm; concentration of all anions, 8 x mollL in water; separation potential, -30 k V other conditions as for Figure 2. Peak identification, (1) I-, (2) SCN-, (3) NOS-, (4) Br-, (5) NOn-, (6) N3-, (7)CI-, (8)F-, (9) C ~ 0 4 ~(10) - , S Z O ~ ~and - , (11)SOP-.
order was observed in water. Changes in solvation of the ions are likely a major factor determining these observed changes in separation selectivity. Ions having different hydrophobicity will have different effective ionic radii in the aqueous and nonaqueous solvents, which will lead to differences in their relative migration orders. During these investigations, significant changes in selectivity were also observed for a given solvent when the nature or concentration of the electrolyte was changed. Ion association, which appeared to be consistent with some aspects of results discussed for Figures 1and 3, is most likely responsiblefor these selectivity changes. In nonaqueous solvents ion association can be extensive, and although considerable information is known about specific ion/solvent systems, the prediction of ion association is dif6cult, especially in mixed-solvent systems where specific ion/solvent interactions can cause significant changes in ion/ion interaction^.^^ For a solvent such as methanol, which is capable of hydrogen bonding, changes in ion association as a function of size of the crystal radii of a series of ions such as tetrapropyl- and (30)Popovych, 0.; Tomkins, R P.T. " a q u e o u s Solution Chemistty;John Wdey & Sons: Toronto. 1981.
Figure 7. Separation of inorganic anions in dimethylformamide: (A) electrolyte, 0.05 mol/L TEAP; (B) electrolyte, 0.1 moVL TBAHP; detection potential, +1700 mV vs SCE. Sample concentrations: ( 8 ) SCN- and N3-, 5 x 10-6 mol/L, I- and NOZ-, 1 x loW5molR, and CI- 1 x 1 0-4 mol/L; (A) all anions, 1 x 10-5 mol/L; peak identification as for Figure 6; other conditions as for Figure 3.
tetrabutylammonium or iodide and bromide, follow trends that are similar to those seen in water.% Thus, in a chromate/ methanol system, which contained mainly K+ (0.01 mol/L) counterions, Br- eluted before NOS-, but in an electrolyte that also contained 0.01 mol/L nbutylammonium cation (Figure 6), Br- eluted after NOS-. In aprotic solvents, such as dimethylformamide, significantly different trends in ion association as a function of nature of the cation and anion are expected.% Evidence of this can be seen in Figure 7 4 where separation in dimethylformamide produces significantly different separation orders relative to 98%methanol (note that the peak numbers in Figure 7 refer to the separation order obtained in 98%methanol in Figure 1). Changes in ion association as a function of the nature and concentration of the electrolyte are illustrated by the reversal of the separation of NOz- and I- from Figure 7A to B when the electrolyte is changed from TBAHP to TEAP. Extensive ion association would be expected to reduce the potential differentiation between analytes, but this may not be a serious problem for most analytes because the extent and trends for ion association can be changed drastically via changes in the properties of the nonaqueous solvent.30 For the systems studied here, the extent of ion association appears to be in the 10-20% range. This range is suggested by the fact that, relative to an aqueous electrolyte, the sensitivity of indirect UV detection (see below) was decreased by -16% in methanol (molar absorbances of the indirect detection reagent were essentially the same in both methanol and water). Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
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Indirect W Detection. Indirect W detection in CE, which was first reported by Hjerten et al.,3l has been a useful technique during the initial development of CE. Because a wide variety of test solutes can be detected with reasonable sensitivity, the application of indirect UV detection was examined briefly for monitoring the migration behavior of the test solutes. The chromate, phthalate, and benzoate reagents were chosen because they are commonly used in water systems. Studies with water added to methanol showed no sudden or erratic CE behavior as a function of water content in the 1-2% range. Consequently, the solvents used in these studies normally contained 1-2% (v/ v) water because this simplified preparation of concentrated stock solutions of the electrolytes. Samples were injected dissolved either in the methanol electrolyte or in water. Good results were obtained with aqueous samples for all nonaqueous systems because the direction of the electroosmotic flow was counter to the sample ion movement, and upon application of the separation potential, the water was removed from the capillary. Indirect response signals were obtained with all reagents. The sensitivity of Cr042-was slightly higher than that for the other reagents, but background noise increased with time due to a slow oxidation/reduction reaction between Cr042- and methanol; however, chromate may be suitable for other less easily oxidized organic solvents. Benzoate was the least sensitive reagent and thus was not studied in detail. Phthalate electrolytes exhibited slightly poorer sensitivitythan CQ- (-50%) but gave reasonably stable baselines and acceptable peak-to peak noise. An electrcpherogram of the separation of 11 anions at concentrations that were 2-3 times the detection limits with phthalate in methanol is shown in Figure 6; detection limits were -3 x mol/L for the monovalent ions in this separation. Sensitivity plots for peak areas showed maximum ranges of change in response factors from 4%for NOz- to 55%for NO3- in the concentration range of 8 x 4x mol/L; correlation coefficients were between 0.997 and 0.999 for all anions in both chromate and phthalate systems. In general, peak shapes were good and separation efficiencies were in the range of 92 000-212 000 for KHP and 100 000370 000 for Cr042- (separation potential -3OkV). Electrochemical Detection. The application of electrochemical detection at ultramicroelectrodes is attractive for CE.32-35 Ultramicroelectrodes have small detection volumes, high sensitivity, and low cost. A further advantage may be realized if organic solvents are used because of the possibility of having wider working potential ranges. Studies with Pt ultramicroelectrodes gave good peak shapes with efficiencies in the range of 140 000450 000 theoretical plates (see Table 1). The choice of electrolyte did affect peak widths, as is illustrated by the results in Figure 7. In Figure 7 4 the separation of NOz-, I-, and Br- with a TBAHP electrolyte in dimethylformamideproduced peak heights of 3-4 nA, while in a TEAP electrolyte, as shown in Figure 7B, peak heights are 2-3 times higher. These differences were due to an decrease in separation efficiency in the TBAHP electrolyte, possibly as a result of ion association equilibria. (31) Hjerten, S.; Elenbring, IC; Kilar,F.; Liao, J. L.; Chen, A. J. C.; Siebert, C. J.; Zhu, M. D. J. Chromatogr. 1987,403, 47-61. (32) Lu, W.; Cassidy, R M. Anal. Chem. 1993,65, 1649-1653. (33) Lu, W.; Cassidy, R M. Anal. Chem. 1993,65, 2878-2881. (34) Lu, W.; Cassidy, R M. Anal. Chem. 1994,66, 200-204. (35) Curry, P. D.; Engstrom-Silverman,C. E.; Ewing, A. G. Electroanalysis 1991, 3, 587-596.
1072 Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
Table 1. Detection Limits for Electrochemical Detection at 25 pm Pt Electrode.
detection limit (mol/L, S/N = 2)
anion SCN-
4
N3-
1
no. of theoretical plates 450 000 250 OOO 280 000 140 000
10-9 10-9 6 x lo-*
INOz-
5x
10-8
Experimental conditions: separation voltage -30 kV, capillary length 50 cm, detection potential +1700 mV, background electrolyte 0.05 mol/L tetraethylammonium perchlorate, 0.1 mol/L n-BuNH2, and 20% (v/v) acetonitrile in dimethylformamide; samples were injected by electromigration at -1 kV for 15 s. 40 h
0 0
1E-M
2E-06
3E-06
Concentration (mom) Figure 8. Calibration curves of SCN- and NB- in dimethylformamide. electrolyte, 0.05 mollL TEAP; detection potential +1700 mV vs SCE; separation voltage, -30 kV; other conditions as for Figure 3.
With an applied detection voltage of +1700 mV, very good detection limits were observed for some anions, as is shown in Table 1 for 0.05 mol/L TEAP in dimethylformamide as the separation electrolyte. These detection limits, which are as low as 1 x mol/L, are considerably smaller than those normally observed in our laboratory under similar experimental conditions with aqueous media20 (Le., lO-'-lO-5 mol/L), Unfortunately, calibration curves (peak area and peak height) were linear only over small concentration ranges. Figure 8 gives results for SCNand N3- (samples dissolved in dimethylformamide), and it can be seen that the calibration curves exhibited linearity only in the concentration range between 5 x and 1 x mol/L for SCN- and 5 x and 5 x loW7mol/L for N3-. Since this nonlinear behavior could have been caused by changes of the surface of the working electrode, pulsed amperometric detection was employed in an attempt to clean the electrode surface prior to each measurement. Peak response increased with the application of a square-wave pulsed signal (1700 mV for 44 ms and a 0 mV for 16 ms),but calibration curves were still nonlinear. When the calibration curve for azide was determined by indirect W detection under similar conditions for the concentration range 10-6-10-3 mol/L, it was found that the calibration curve was still nonlinear. Thus, for azide a possible explanation for the nonlinearity may be due decomposition or strong ion association of these anions in dimethylformamide. Additional studies are needed to determine the exact cause of these problems. CONCLUSIONS
The results in this study suggest that one of the most attractive features of nonaqueous CE systems may be the ability to easily
vary selectivity via changes in the nature of the solvent and the nature and concentration of the electrolyte. Recent results in our laboratory on the development of methods for the CE determination of straightchain alkanesulfonates and sulfates also show significantly reduced peak broadening (sorption) and solubility problems for chain lengths in the ClO-cl8 range. As there is a large amount of information available about ion/ion and ion/ solvent interactions in the literat~re,~O nonaqueous systems may prove to useful for a number of analysis problems, but further controlled studies are needed to develop a more comprehensive understanding of the behavior of different electrolyte/solvent systems. Detection with electrochemical techniques also appears promising, but considerable work is required to adequately define
proper experimental conditions to improve reproducibility and linearity. ACKNOWLEDQMENT We acknowledge the hancial support of the Natural Science and Engineering Research Council of Canada and the Waters Corp. Received for review October 7, 1994. Accepted December 19, 1994." AC940992P Abstract published in Advance ACS Abstracts, February 1, 1995.
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